[The NS gene--a possible determinant of apathogenicity of a cold-adapted donor of attenuation A /Leningrad/134/47/57 and its reassortants]. / Gen NS--veroiatnaia determinant apatogennosti kholodoadaptirovannogo donora attenuatsii A /Leningrad/134/47/57 i ego reassortantov.

Phenotypical properties of single-gene reassortants of attenuated cold-adapted strain A/Leningrad/135/47/57 (H2N2) and strain A/PR8/34 virulent for laboratory animals were studied. Only the group of reassortants inheriting NS gene from cold-adapted virus was fully attenuated for various animals species, similarly as reassortants with 6/2 genomic formula containing all the 6 internal protein genes from strain A/Leningrad/134/47/57. Reassortant 25A-1 single-gene for NS was temperature-sensitive (ts) on mammalian cells but formed plaques at 40 degrees C on chicken kidney cells. Reassortants with genomic formula 6/2 were temperature-sensitive in all types of cells used. Reassortant 25A-1 could synthesize normal amounts of polypeptides in MDCK cells at 39 degrees C, whereas protein synthesis of reassortants with 6/2 genomic formula was noticeably reduced at this temperature. Hence, a similar level of attenuation of both reassortant groups appears to be due to various molecular mechanisms. Possible role of NS2 gene mutation in attenuation of strain A/Leningrad/134/47/57 and its reassortants is discussed.

[Immunogenicity of attenuated reassortants of influenza A virus /Leningrad/134/47/57 (H2N2) for mice, depending on their genome]. / Immunogennost' dlia myshei attenuirovannykh reassortantov virusa grippa A /Leningrad/134/47/57 (H2N2) v zavisimosti ot sostava ikh genoma.

Reassortants of cold-adapted strain A/Leningrad/134/47/57 (H2N2) and virulent strain A/PR/8/34 (H1N1), modeling genomic composition of vaccinal strains, were tried in mouse experiments. Reassortants' genomes included the major part (5, 6, or 7) from cold-adapted strain and 1 to 3 genes of virulent strain. All the tested reassortants did not differ by temperature sensitivity or cold adaptation phenotypes, did not cause the death of mice, but differed by the level of reproduction in murine lungs and by immunogenicity. Strains with genomic formulas 6/2 (HA and NA of a virulent strain) and 5/3 (M) (HA, NA, and M of a virulent strain) were characterized by the highest immunogenicity. Reassortants including, besides HA and NA, PA gene 5/3 (PA) or inheriting only HA gene from strain A/pr/8/34 (H1N1) were hyperattenuated and low immunogenic.

[Variations in the temperature range for reproduction of virulent and attenuated cold-adapted influenza A viruses]. / Razlichiia v temperaturnom diapazone reproduktsii virulentnykh i attenuirovannykh kholodoadaptirovannykh virusov grippa A.

The informativity of RCT40, RCT37.5, and RCT25 markers for differentiation of wild type strains from ca-recombinants obtained on the basis of ca donors of attenuation was studied. The RCT25 marker and express RCT37.5 marker (determined within 24 hours) were found to be universal characteristics differentiating ca-recombinants from virulent viruses.

Study of live recombinant cold-adapted influenza bivalent vaccine of type A for use in children: an epidemiological control trial.

Live cold-adapted recombinant bivalent vaccine of influenza type A was studied in a controlled field trial in 1982-1983 among nearly 30,000 children 3-15 years old. The bivalent vaccine consisted of recombinants 47/25/1 (H1N1) and 47/7/2 (H3N2) of wild-type viruses A/Brazil/11/78 (H1N1) and A/Bangkok/1/79 (H3N2) with cold-adapted donor A/Leningrad/134/47/57 (H2N2). The recombinants which received mutant nonglycoprotein genes from cold-adapted donor did not suppress each other after simultaneous inoculation of children and stimulated antibody response to both strains. The bivalent vaccine was completely attenuated for children. It caused less than 1% transient febrile reactions during five days after the first vaccination, including double seronegative individuals with low antibody titres to both vaccinal strains. The cold-adapted bivalent vaccine tested proved to be safe for children according to the analysis of morbidity studies among vaccines and a control group performed during the five days and the following six months after the first immunization. There is a similar distribution of non-influenza illnesses and a statistically significant decrease in influenza-like diseases among vaccines compared to the control group. In the four months after the immunization programme was completed, epidemics of influenza A H1N1 and H3N2 occurred. The incidence of influenza-like diseases was approximately 50% less in the vaccinated than in the control groups. This is the first evidence of safety and protective efficacy of recombinant live influenza vaccine for children 3-15 years of age.

[Characteristics of incomplete reproduction cycle of ts-mutant of influenza virus A at non-permissive temperature]. / Kharakteristika nezavershennogo tsikla reproduktsii ts-mutanta virusa grippa A pri nerazreshaiushchei temperature.

Incomplete reproduction cycle of influenza virus A/134/17/57, the attenuation donor being used for preparation of recombinant vaccine strains, hs been analyzed with the use of molecular biology methods. Virus A/134/17/57 with two mutations in P3, NP and M genes remains capable of synthesis of viral polypeptides that are devoid of ability to be inserted into cellular plasma membrane, when the virus is propagated in MDCK culture at non-permissive temperature. The process is preceded by defects in the process of formation of RNP structures connected, evidently, with deficient synthesis of viral RNA due to mutations in the gene coding for P3 protein.

Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees.

Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of vaccines at a 3-week interval, both strains induced antibody in 75 to 95% of the children. On coinfection of chicken embryos with both recombinants, only weak interference was observed. Administration to children of the bivalent vaccine containing H1N1 and H3N2 recombinants induced efficient production of antibody to H1 and H3 hemagglutinins and N1 and N2 neuraminidases without adverse reactions. The recombinants studied were genetically stable as judged by retention of the temperature-sensitive phenotypes and a lack of reversion of the genes carrying temperature-sensitive mutations in all of the reisolates from vaccinated children.

Location of ts defects in the genome of cold-adapted recombinant influenza A virus vaccine strains.

The ts phenotype and location of ts mutations were studied in the genome of parent viruses and those obtained by recombination of cold-adapted strains A/Leningrad/134/17/57 or A/Leningrad/134/47/57 with epidemic H1N1 and H3N2 influenza A virus strains. The epidemic H1N1 and H3N2 strains under study possessed a ts phenotype and contained ts mutations in one or two genes. The ts phenotype was lost following three clonings at 40 degrees C, suggesting that influenza virus strains isolated from humans may be heterogeneous and contain virions either carrying or not carrying the ts mutations in their genomes. Two cold-adapted strains possessing a distinct ts phenotype contained ts mutations in three (A/Leningrad/134/17/57 virus after 17 passages at 25 degrees C) or in five (A/Leningrad/134/47/57 variant after 30 additional passages at 25 degrees C) genes coding for non-glycosylated proteins. When compared with cold-adapted donor strains, the recombinants had either the same set or additional ts mutations. However, no ts mutation was detected in a gene which had been inherited from the donor strain. It is suggested that, in addition to the analysis of the genome composition, in cold-adapted recombinant influenza virus strains recommended as vaccine candidates it is necessary to control the number of genes containing ts mutations.

[Cold-adapted strain of A/Leningrad/134/47/57 (H2N2)--a special attenuation donor of live influenza vaccine for children and the isolation of its recombinants]. / Kholodoadaptirovannyi shtamm A/Leningrad/134/47/57 (H2N2)--spetsialnyi donor attenuatsii zhivoi grikpoznoi vaktsiny dlia detei i poluchenie na ego osnove rekombinantov.

A cold-adapted A/Leningrad/134/47/57 (H2N2) strain, a special donor of attenuation for generation of recombinant vaccine strains safe for children, has been developed and characterized. The genetic markers of this virus (temperature sensitivity of the cold-adapted strain, temperature optimum of the neuraminidase activity) were compared with those of A/Leningrad/134/17/57 (H2N2) strain, a donor of attenuation for a live influenza vaccine for adults. The A/Leningrad/134/47/57 (H2N2) strain was found to have a more marked temperature sensitivity and cold adaptation which correlated with the presence in its genome of multiple temperature-sensitive mutations in genes 1, 2, 5, 7, and 8. When the cold-adapted A/Leningrad/134/47/57 (H2N2) strain was hybridized with epidemic A/Brazil/11/78 (H1N1) and A/Bangkok/1/79 (H3N2) strains, recombinants were isolated which are candidate vaccine strains for a live influenza vaccine for children. The recombinants inherited the antigenic specificity of the current viruses and the temperature specificity and cold-adaptation of the donor of attenuation, A/Leningrad/134/47/57 (H2N2).

[Safety and high immunogenicity for children 3 to 15 of a mono- and a divaccine from recombinant influenza A/Brazil /11/78 (H1N1) and A/Bangkok/1/79 (H3N2) viruses obtained from a special donor attenuation base--the cold-adapted virus A/Leningrad/134/47/57 (H2N2)]. / Bezvrednost' i vysokaia immunogennost' dlia detei 3--15 let mono- i divaktsiny iz rekombinantov virusov grippa A/Braziliia/11/78 (H1N1) i A/Bangkok/1/79 (H3N2), poluchennykh na osnove spetsial'nogo donora attenuatsii--kholodoadaptirovannogo virusa A/Leningrad/134/47/57 (H2N2).

The data on complete safety and high immunogenic potency of intranasal live influenza vaccine from the recombinant A/Brazil/11/78 (H1N1) and A/Bangkok/1/79 (H3N2) strains prepared with a special donor of attenuation, a cold-adapted A/Leningrad/134/47/57 (H2N2) virus, have been obtained. The possibility of using a bivaccine from the recombinant strains constructed on the basis of a single donor of attenuation and inheriting from it most of the genes coding for nonglycosylated proteins was demonstrated. Administration of this bivaccine to children was not accompanied by any increase in the rate of postvaccination febrile reactions, but produced in the vaccinees an intensive immune response to the antigenic components of the vaccine which was in agreement with the data obtained after separate administration of monovaccines.

Attenuated ts-recombinants of influenza A/USSR/77 (H1N1) virus obtained by crossing with the cold-adapted donor A/Leningrad/134/57 (H2N2) virus.

Conditions of obtaining attenuated influenza virus recombinants by crossing of a cold-adapted donor with A (H1N1) influenza virus that reappeared in 1977 were studied. Temperature-sensitive recombinants suitable for intranasal immunization of adults with low titres of anti-haemagglutinin and anti-neuraminidase antibodies, and possessing sufficiently high immunogenicity were obtained by crossing of native parent strains and cross-reactivation techniques. It was confirmed that the cold-adapted A/Leningrad/134/17/57 (H2N2) influenza virus variant is a prospective donor of attenuation for obtaining recombinants--candidates for live influenza vaccine strains.

Partial change of the ts-phenotype of cold-adapted influenza virus strain grown in canine kidney cells in the presence of trypsin.

The cold-adapted temperature-sensitive (ts) influenza virus strain A/Leningrad/134/17/57 (H2N2) multiplied well at 32 degrees C (optimal temperature); lower titres of infectious virus were obtained in developing chick embryos at 40 degrees C. In a canine kidney (MDCK) cell line and in primary calf kidney (CK) cells an increased reproduction of the virus was found at 40 degrees C especially in the presence of trypsin. The ratios of virus titres obtained at optimal versus higher temperatures (RCT40) were by 1,000 times lower than those found in chick embryos. Polyacrylamide gel electrophoresis revealed a comparable synthesis of the cold-adapted influenza virus strain polypeptides HA, NP, M and NS in MDCK cells, regardless whether they were incubated at optimal or non-permissive temperatures.

Analysis of ts mutations of cold-adapted influenza A/Leningrad/134/57 virus variants.

By recombination of ts mutants of fowl plague virus belonging to different complementation groups with two cold-adapted variants of human influenza virus, the number and gene localization of ts mutations occurring in these variants was determined. In the course of passaging of human influenza virus at lowered temperature, the number of genes with ts mutations increased.

Peculiarities of obtaining attenuated thermosensitive recombinants of influenza A virus at the end of the H3N2 epidemic cycle.

The conditions of obtaining thermosensitive recombinants of the virulent A/Leningrad/82/76 (H3N2) virus with two donors of attenuation, A/Leningrad/134/17/57 (H2N2) and A/Leningrad/9/37/46 (H0N1), were evaluated. The recombinants were obtained by various methodical approaches (hybridization of native viruses, cross-reaction and selection of recombinants at temperatures of 25, 32 and 40 degrees C) to study their effects on the degree of attenuation and the regularity of transmission of the ts marker. The recombinants examined varied in thermosensitivity which depended on the conditions of recombination of parent viruses. All recombinants studied, irrespective of their RCT40 marker, were innocuous for men.

[The effect of thymosine on the course of experimental influenza infection and antibody production in laboratory animals]. / Vliianie timozina na techenie éksperimental'noi grippoznoi infektsii i protsess antiteloobrazovaniia u laboratornykh zhivotnykh.

Thymosine is a preparation isolated from the thymus of calves. It possesses a specific activity stimulating maturation of T lymphocytes in splenocytes of thymectomized guinea pigs and peripheral blood of healthy humans. Thymosine had no effect on the course of severe experimental influenza infection and exerted no regular stimulation of antibody production in rats with asymptomatic influenza infection.

[Experience in selecting safe heat-sensitive influenza virus A recombinants]. / Opyt selektsii bezvrednykh termochuvstvitel'nykh rekombinantov virusa grippa A.

Conditions for genetic recombination and laboratory testing of influenza A virus for rapid production of safe vaccine strains needed in human vaccination were developed. The best donors of attenuation were found to include limitingly attenuated outdated vaccine strains adapted to low temperature and having important differentiating features such as thermosensitivity and reduced optimal temperature of neuraminidase activity. Trials in volunteers of a large collection of recombinants produced by recombination of these donors with various virulent strains circulating in 1972--1976 (A/Victoria/72, A/Port Chalmers/73, and A/Virtoria/75) demonstrated complete safety for man of clones with different combinations of rct/40 and OTNA genetic markers.

Conditions for production of thermosensitive attenuated influenza virus recombinants.

Recombination and cross-reactivation between virulent influenza viruses and a cold-adapted thermosensitive vaccine strain regularly produced genetically stable attenuated recombinants, the selection of which was based on the thermosensitivity marker. This marker, correlating with the safety of the recombinants for man was inherited independently on the properties of the haemagglutinin and neuraminidase surface antigens. There was no relationship between the thermosensitivity of the resulting recombinants and the decrease in the optimal temperature of the neuraminidase activity (OTNA marker). This indicates a separate localization in the viral genome of the mutation damages responsible for the expression of ts and OTNA markers.